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题名	固斑蝰蛇毒C-型凝集素样蛋白Dabocetin及L-氨基酸氧化酶DRS-LAO的研究
作者	钟树荣
学位类别	博士
答辩日期	2005-06
授予单位	中国科学院研究生院
授予地点	北京
导师	熊郁良 ; 张云
关键词	圆斑蛙蛇 蛇毒C-型凝集素 GP几结合蛋白 L一氨基酸氧化酶 分子克隆
其他题名	Studies on a C-type lectin-like protein (dabocetin) and an L-amino acid oxidase (DRS-LAO) from Daboia russellii siamensis
中文摘要	本论文研究了从圆斑蛙蛇泰国亚种（Daboia russellii siamensis）蛇毒中纯化的C一型凝集素样蛋白Dabocetin和L一氨基酸氧化酶DRS一AO的理化性质、生物学活性和分子克隆。Dabocetin是分子量约为28扔。的异二聚体蛋白，它由分子量约为15.0kDa和14．5kDa的两个同源亚基以和p共价结合形成。N－末端氨基酸序列比较显示，Dabocetin与目前已知的蛇毒c一型凝集素样蛋白有很高的同源性。即使在终浓度达50．0。叫而时，Dabocetin也不能直接诱导血小板聚集。此外，在终浓度为40.00μg/ml时，Dabocetin几乎不能抑制由AdP，TMVA和stejnulxin诱导的血小板聚集。但是，Dabocetin呈剂量依赖地抑制瑞斯托霉素诱导的血小板凝集，其半数抑制率ICS。值为10.80ug/ml。流式细胞仪分析表明，Dabocetin显著抑制单克隆抗体522与GPIba的结合，提示Dabocetin很可能是一个GPIb结合蛋白。从圆斑蛙蛇的毒腺中克隆到了7个编码不同蛇毒C一型凝集素样蛋白亚基的七DNA（命名为DRs一1至DRs一7）。其中，DRsLS编码Dabocetin的a亚基，DRS一6编码Dabocetin的p亚基。DRs一1和DRS一2很可能是圆斑蛙蛇毒腺中表达的X因子激活剂的两条轻链LCZ和LCI的山NA。DRS一3，DRS毛4和DRSL7可能是圆斑蛙蛇毒腺中表达的C一型凝集素样蛋白p亚基的。NA。DRsLAO是一个新的L一氨基酸氧化酶，比活力为1．98U加噶。十二烷基磺酸钠一聚丙烯酞氨凝胶电泳（SDs-PAGE）分析显示，该酶在还原和非还原条件下均呈现一条蛋白带，表观分子量约为58kDa。N-末端氨基酸序列比较显示，DRS一AO与目前已知的蛇毒L一氨基酸氧化酶有很高的同源性。该酶的最适底物为L一亮氨酸，最适pH为8．8。DRs一Ao呈剂量依赖地抑制扔P和仆IvA诱导的血小板聚集，其半数抑制率ICS。值分别为32.8μg/ml和32.3μg/ml。DRS-LAO对金黄色葡萄球菌（灯Cc25923）和耐甲氧西林金黄色葡萄球菌有较强的抗菌作用。DRs一AO对金黄色葡萄球菌必Tcc25923）的最低抑菌浓度卿C）和最低杀菌浓度耐甲氧西林金黄色葡萄球菌的孤CS。和呱Cg。值分别为18．。林留时和36.0μg/ml；DRSLAo对耐甲氧西林金黄色葡萄球菌的MBC50和MBCg。值分别为36.0μg/ml和72.0μg/ml。通过对DRS一AO的分子克隆，得到了编码DRS-AO的部分cDNA序列。
英文摘要	Studies on physical and chemical properties, biological activities and molecular cloning of a novel C-type lectin-like protein, named Dabocetin and an L-amino acid oxidase, named DRS-LAO, from the venom of Daboia russellii siamensis were presented in this thesis. Dabocetin is a 28 kDa hetermeric protein consisted of two distinct but homologous subunits linked by disulfide bond. The apparent molecular weights of a and p subunits were 15.0 kDa and 14.5 kDa, respectively, when estimated by SDS-PAGE under reducing conditions. Comparison of the N-terminal amino acid sequences of Dabocetin a and p subunits with those of other known snake venom C-type lectin-like proteins showed that Dabocetin is homologous to other known snake venom C-type lectin-like proteins. Dabocetin did not induce direct platelet aggregation in platelet-rich plasma even at a final concentration of 50.00 ug/ml. Moreover, it has little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin at the dosage of 40.00 ug/ml. Whereas Dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC50 value of 10.80 ug/ml. Flow cytometry analysis showed that Dabocetin significantly inhibit mAb SZ2 binding to platelet membrane glycoprotein Ib alpha. This indicated that Dabocetin is probably a GPIb-binding protein. Seven cDNAs encoding various snake venom C-type lectin-like protein precursors (designated as DRS-Ll to DRS-L7), were obtained from a venom gland of snake Daboia russellii siamensis. DRS-L5 and DRS-L6 encoded Dabocetin a and p subunits, respectively. DRS-Ll and DRS-L2 are probably cDNAs of the light chain LC2 and LCI of factor X activator expressed in Daboia russellii siamensis venom gland, respectively. DRS-L3, DRS-L4 and DRS-L7 might be P subunits of snake venom C-type lectin-like proteins expressed in Daboia russellii siamensis venom gland. DRS-LAO is a novel L-amino acid oxidase and its specific activity is determined to be 1.98 U/mg. It was homogeneous with an apparent molecular weight of 58 kDa both under non-reducing and reducing conditions of SDS-PAGE. Comparison of the N-terminal amino acid sequences of DRS-LAO with those of other known snake venom L-amino acid oxidase showed that DRS-LAO is homologous to the reported snake venom L-amino acid oxidases previously. The optimum substrate and pH are L-leucine and pH 8.8, respectively. Dabocetin inhibited platelet aggregation induced by ADP and TMVA in platelet-rich plasma in a dose-dependent manner with an IC50 value of 32.8 ug/ml and 32.3 fig/ml, respectively. DRS-LAO has strong activity against Staphylococci aureus (ATCC 25923) and methicillin-resistant Staphylococcus aureus. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of DRS-LAO against Staphylococci aureus (ATCC 25923) were 9.0 ug/ml and 18.0 ug/ml, respectively. TheMIC5o andMICso of DRS-LAO against methicillin-resistant Staphylococcus aureus were 18.0 ug/ml and 36.0 ug/ml, respectively. The MBC50 and MBC90 of DRS-LAO against methicillin-resistant Staphylococcus aureus were 36.0 ug/ml and 72.0 ug/ml, respectively. Partial cDNA encoding DRS-LAO was obtained from a venom gland of snake Daboia russellii siamensis.
语种	中文
公开日期	2010-10-15
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6169]
专题	昆明动物研究所_其他 昆明动物研究所_动物活性蛋白多肽组学
作者单位	中国科学院昆明动物研究所
推荐引用方式 GB/T 7714	钟树荣. 固斑蝰蛇毒C-型凝集素样蛋白Dabocetin及L-氨基酸氧化酶DRS-LAO的研究[D]. 北京. 中国科学院研究生院. 2005.

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